Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis

Abstract

Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into ‘targetrons.’ Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and ‘cut-and-pastes’ (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The high processivity and fidelity of group II intron reverse transcriptases along with their novel template-switching activity, which can directly link RNA-seq adaptor sequences to cDNAs during reverse transcription, open new approaches for RNA-seq and the identification and profiling of non-coding RNAs, with potentially wide applications in research and biotechnology.

Characterization of cis -Acting RNA Elements of Zika Virus by Using a Self-Splicing Ribozyme-Dependent Infectious Clone

“…Thus, these eukaryotic intron-based infectious clones are not applicable for viral replication studies. In addition, the intron-based reverse genetics system retained the potential to convert into in vivo splicing-based systems since the self-splicing reaction can be facilitated by the RNA maturase activity of IEP in vivo (30,31,53), which can be supplied in trans.…”

High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases

“…Mobile group II introns are bacterial retrotransposons that proliferate within genomes by a mechanism called retrohoming, which requires reverse transcription of a highly structured group II intron RNA with high processivity and fidelity, properties that are potentially useful for applications that require cDNA synthesis, such as RNAseq and qRT-PCR (Mohr et al 2013;Enyeart et al 2014;Lambowitz and Belfort 2015). Recently, we overcame a long-standing impediment by developing general methods for the high-level expression of thermostable group II intron RTs (TGIRTs) from bacterial thermophiles as fusion proteins with a noncleavable solubility tag attached via a rigid linker (Mohr et al 2013).…”

A Coming of Age Story: Chlamydia in the Post-Genetic Era

“…Additionally, failure to generate a mutant could result from improper transformation/intron function or polar effects acting on an essential gene, rather than the essentiality of the targeted gene. The Ll.LtrB group II intron has recently been modified to allow for Cre-mediated recombination, which could be adapted for genome editing or marker recycling, and a second intron-based method using an EcI5 intron has been developed that is more efficient than the LtrB intron (not evaluated in Chlamydia) (74). The TargeTron system has also been used in the obligate intracellular pathogens Rickettsia rickettsii and Ehrlichia chaffeensis (77,78).…”

“…The required mutations are identified using a proprietary algorithm. With the assistance of an intron-encoded protein, LtrA (expressed independently from the intron), the intron is spliced into the target gene creating an insertional gene mutant (74). The intron is introduced into C. trachomatis on a suicide vector, and selection of insertion is performed using ampicillin (the intron carries a bla marker).…”